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Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

journa l h om epage: www.elsev ier .com/ locate /co lsur fa

ovine serum albumin (BSA) and cleaved-BSA conjugated ultrasmalld2O3 nanoparticles: Synthesis, characterization, and application toRI contrast agents

d. Wasi Ahmada, Cho Rong Kima, Jong Su Baeckb, Yongmin Changb,d,∗,ae Jeong Kimc,d, Ji Eun Baed, Kwon Seok Chaed,e, Gang Ho Leea,d,∗

Department of Chemistry, College of Natural Sciences, Kyungpook National University (KNU), Taegu 702-701, South KoreaDepartment of Molecular Medicine and Medical & Biological Engineering, School of Medicine, KNU, Taegu 702-701, South KoreaDepartment of Applied Chemistry, College of Engineering, KNU, Taegu 702-701, South KoreaDepartment of Nanoscience and Nanotechnology, KNU, Taegu 702-701, South KoreaDepartment of Biology Education, Teacher’s College, KNU, Taegu 702-701, South Korea

i g h l i g h t s

BSA and C-BSA conjugated ultrasmallGd2O3 nanoparticles were synthe-sized.BSA can bind many ultrasmall Gd2O3

nanoparticles whereas C-BSA cannot.Large water proton relaxivities wereobserved.High contrast MR images in a mouseliver after intravenous injection wereobserved.These nanoparticles are potentialMRI contrast agents.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 15 November 2013eceived in revised form 23 February 2014ccepted 1 March 2014vailable online 12 March 2014

eywords:

a b s t r a c t

Bovine serum albumin (BSA) (Mn = 66.5 kD, size = 14 × 4 × 4 nm) is an attractive biological molecule forbiomedical applications because of its water-solubility and bio-compatibility. It can also bind manyultrasmall nanoparticles (NPs) as confirmed in this study. We synthesized polyethylene glycol diacid(PEGD) coated ultrasmall Gd2O3 nanoparticles (PEGD-GNPs, the core davg = 2.0 nm), which were then con-jugated to BSA and cleaved-BSA (C-BSA) (i.e. BSA-PEGD-GNPs and C-BSA-PEGD-GNPs) through amidebonding. Large relaxivities were observed in both aqueous sample solutions (r1 = 6.0 s−1 mM−1 and

−1 −1 −1 −1 −1 −1

SA-BSAltrasmall Gd2O3 nanoparticleanoparticle carrierRI

ontrast agent

r2 = 28.0 s mM for BSA-PEGD-GNPs and r1 = 7.6 s mM and r2 = 22.0 s mM for C-BSA-PEGD-GNPs). Three tesla T2 magnetic resonance imaging (MRI) in a mouse after the injection of an aqueoussample solution of BSA-PEGD-Gments. Large relaxivities and ipotential MRI contrast agents.

Abbreviations: BSA, bovine serum albumin; C-BSA, cleaved BSA; PEGD, polyethylene ghosphate buffer solution; MRI, magnetic resonance imaging; NCT, neutron capture theraphosphate buffer solution; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminlectron microscope; XRD, X-ray diffraction; ICPAES, inductively coupled plasma atomic enalyzer; DU145, human prostate cancer cell; NCTC1469, normal mouse hepatocyte cell.∗ Corresponding authors. Tel.: +82 53 950 5340; fax: +82 53 950 6330.

E-mail addresses: ychang@knu.ac.kr (Y. Chang), ghlee@mail.knu.ac.kr (G.H. Lee).

ttp://dx.doi.org/10.1016/j.colsurfa.2014.03.011927-7757/© 2014 Elsevier B.V. All rights reserved.

NPs into a mouse tail vein revealed significant negative contrast enhance-

n vivo MR images prove that BSA-PEGD-GNPs and C-BSA-PEGD-GNPs are

© 2014 Elsevier B.V. All rights reserved.

lycol diacid; NP, nanoparticle; GNP, Gd2O3 NP; BRB, Britton–Robinson buffer; PBS,y; CT, X-ray computed tomography; DTPA, diethylenetriaminepentaacetic acid; PBS,opropyl) carbodiimide; GPC, gel permeation chromatograph; HVEM, high voltage

mission spectrometer; FT-IR, Fourier transform-infrared; TGA, thermo-gravimetric

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8 Md.W. Ahmad et al. / Colloids and Surfaces

. Introduction

Biological molecules have attracted considerable interest forpplication to nanomedicine because they are fully biocompatiblend water-soluble. Bovine serum albumin (BSA) is an importantarrier protein in blood plasma for several ions and molecules. It has

large dimension (14 × 4 × 4 nm) and heavy mass (Mn = ∼66.5 kD).hus, it may also carry several ultrasmall nanoparticles (NPs) with

smaller size and mass than BSA. Biological molecules have sev-ral advantages over small molecules and polymers for biomedicalpplications. First, the water-solubility of surface-modified NPsenerally increases with increasing mass of the ligands [1,2] andhus, biological molecules will provide enhanced water-solubilityor NPs. Second, the NPs conjugated to biological molecules canemain in the blood for a longer duration than free NPs andd-chelates, allowing longer imaging times (so called blood-pool

maging agents) and a higher likelihood of delivering NPs to theargeted areas in a body [3–6].

This study makes use of BSA and ultrasmall Gd2O3 NPs (GNPs)or magnetic resonance imaging (MRI). Ultrasmall GNPs havehown longitudinal (r1) and transverse (r2) water proton relax-vities larger than those of Gd-chelates because of the denseopulation of Gd(III) in NPs [7,8]. Therefore, BSA which could bindeveral ultrasmall GNPs may be useful for MRI. Here, ultrasmallNPs can be also applied to X-ray computed tomography (CT)s CT contrast agents and neutron capture therapy (NCT) as NCTgents because Gd has a large X-ray attenuation power (∼2.5 timestronger than commercial iodine CT contrast agents) [9–12] and aery large neutron capture cross section (∼254,000 barns) [13–16].his implies that BSA conjugated ultrasmall GNPs could be alsoseful for CT and NCT.

Previous studies on MRI using BSA include BSA[Gd-chelates]n

nd BSA-GNPs (d = 20–40 nm) [3–6,10,17]. Enhanced water protonelaxivities have been observed in both systems after conjuga-ion to BSA. In addition, the in vivo application of BSA[Gd-DTPA]n

DTPA = diethylenetriaminepentaacetic acid) showed a longer cir-ulation time in the blood than Gd-DTPA, providing longer MRmaging times in brain tumors and blood vessels in rats [3,4].

This study examined BSA and cleaved BSA (C-BSA) (<7.0 kD) con-ugated ultrasmall GNPs for MRI contrast agents. Here, ultrasmallanoparticles (d < 3 nm) are clinically important because they cane excreted through renal system [18] and therefore, have a highotential for biomedical applications. To conjugate GNPs to BSAnd C-BSA, the GNPs were surface modified with polyethylenegly-ol diacid (PEGD) (Mn = 600) and then conjugated to BSA and C-BSAia amide bonding. The particle sizes, surface modifications, num-er of ultrasmall GNPs conjugated to BSA and C-BSA, water protonelaxivities, cellular toxicities, and in vivo MR images using a mouseere characterized. The BSA conjugated GNPs were further applied

o MRI in a mouse and negative contrast enhancements in 3 T T2 MRmages were clearly observed.

. Experimentals

.1. Chemicals

All chemicals such as GdCl3·xH2O (99.9%), NaOH (>99.9%), tri-thylene glycol (99%), polyethylene glycol diacid (PEGD) (Mn = 600),SA (Mn = ∼66.5 kD), phosphate buffer solution (PBS) (pH = 7.2),Cl (>99%), N-hydroxysuccinimide (NHS) (98%), 1-ethyl-3-(3-

imethylaminopropyl) carbodiimide (EDC) (>97%), boric acid>98%), phosphoric acid (>97%), and acetic acid (98%) were pur-hased from Sigma-Aldrich and used as received. Triply distilledater was used for both washing the products and preparing theRI sample solutions.

ysicochem. Eng. Aspects 450 (2014) 67–75

2.2. Synthesis of PEGD coated ultrasmall GNPs (PEGD-GNPs)

PEGD-coated ultrasmall GNPs were synthesized using one-potprocess (Scheme 1a). Two separate solutions were prepared. Oneis a precursor solution made from 5 mmol of GdCl3·xH2O in 25 mLof triethylene glycol, and the other is a NaOH solution made from15 mmol of NaOH in 10 mL of triethylene glycol. The precursor solu-tion was heated to 60 ◦C with magnetic stirring under atmosphericconditions until the precursor was dissolved completely in triethy-lene glycol. A NaOH solution was added to the precursor solution.The mixed solution was stirred magnetically at 180 ◦C for 4 h. Thesolution temperature was reduced to 80 ◦C and 8 mmol of PEGD wasadded to the solution for surface coating. An excess of PEGD wasused to ensure that only one –COOH group among the two –COOHgroups in PEGD could be used for conjugation to ultrasmall GNPsbecause the other –COOH group should be free for conjugation toBSA (or C-BSA). The solution temperature was again increased to180 ◦C and stirred for an additional 4 h. The solution was cooledto room temperature and transferred to a 1 L beaker containing500 mL of triply distilled water. This was stirred magnetically for10 min and stored for a week or so until the PEGD-coated ultrasmallGNPs settled in a beaker bottom. The supernatant was decantedand the remaining sample solution was washed again with triplydistilled water. This procedure was repeated three times.

2.3. Synthesis of C-BSA

To prepare C-BSA, 10 mmol of l-serine and 10 mmol of l-histidine were dissolved in 300 mL of a Britton–Robinson buffer(BRB) (40 mM phosphate, 40 mM acetate, and 40 mM borate)(pH = 5–6) at 60 ◦C and under atmospheric conditions (Scheme 1b)[19]. Here, a pH of 5–6 of the buffer solution was achieved by addingNaOH slowly to the original buffer solution. After magnetic stirringfor 30 min, 156 mg of BSA was added to the solution, and the solu-tion was stirred magnetically for 36 h at 60 ◦C. After the reactionwas complete, the water was evaporated. This cleavage reactionwas repeated 10 times to obtain enough C-BSA. The masses of C-BSAs were characterized by gel permeation chromatography (GPC)and the result is summarized in Supporting Information. Two majorC-BSAs with masses of 6.67 and 2.01 kD were observed from GPCanalysis. The C-BSA was used without further purification becausethe water-soluble C-BSA could not be separated from other water-soluble reagents used in the reaction. On the other hand, the watersoluble reagents were later removed after conjugation of C-BSAs toPEGD-GNPs through amide bonding.

2.4. Synthesis of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs

BSA-PEGD-GNPs and C-BSA-PEGD-GNPs were synthesizedusing an EDC/NHS coupling method (Scheme 1c) [20,21]. In thisreaction amide bonds were formed between –COOH of PEGD-GNPsand –NH2 of BSA (or C-BSA). The reaction was carried out at roomtemperature and under atmospheric conditions. Solution pH wasfixed at 6.0 by adding 1 mM HCl to the original PBS with pH of 7.2.5 mmol of EDC and 5 mmol of NHS were added to 30 mL of PBS(pH = 6). After magnetic stirring for 30 min, the PEGD-GNPs wereadded to the solution and the solution was stirred magneticallyfor 2 h. 1.5 g of BSA (or C-BSA) was added to the solution at roomtemperature and stirred for an additional 2 h. The product solutionwas transferred to a 1 L beaker containing 500 mL of triply distilledwater. The resulting solution was stirred magnetically for 10 minand stored for a week until BSA-PEGD-GNPs (or C-BSA-PEGD-GNPs)

settled in the bottom of the beaker. The supernatant was decantedand the remaining sample solution was washed again with triplydistilled water. This procedure was repeated three times. Duringthis process, any water-soluble impurities such as solvent and any

Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75 69

s, (b)

opfde

2

wANmtgwtd

Scheme 1. Syntheses of (a) the ultrasmall GNPs and PEGD-GNP

ther reagents used in synthesis of C-BSA, were removed from theroducts. Portions of the samples were evaporated to a powderorm in air and the remaining portions were diluted with triplyistilled water to prepare aqueous sample solutions for the MRIxperiments.

.5. General characterization

The particle diameters of the ultrasmall GNPs were measuredith a high voltage electron microscope (HVEM) (JEOL JEM-RM1300S, 1.2 MeV acceleration voltage). A copper grid (PELCOo. 160, TED PELLA, INC.) covered with an amorphous carbonembrane was placed onto a filter paper and a sample solu-

ion diluted in triply distilled water was dropped onto the copper

rid using a micropipette (Eppendorf, 2–20 �L). The copper gridas dried in air for one hour to remove the solvent. The crys-

al structure of ultrasmall GNPs was examined using an X-rayiffraction (XRD) spectrometer (Philips, X-PERT PRO MRD) with

the C-BSA, and (c) the BSA-PEGD-GNPs and C-BSA-PEGD-GNPs.

unfiltered Cu-K� radiation of 1.54184 A, scanning step of 0.033◦,and scan range of 2� = 15–100◦. The concentration of Gd in the sam-ple solution was determined using an inductively coupled plasmaatomic emission spectrometer (ICPAES) (Thermo Jarrell Ash Co.,IRIS/AP). The surface coating of the ultrasmall GNPs with PEGDand conjugation of PEGD-GNPs to BSA (or C-BSA) were investigatedusing a Fourier transform-infrared (FT-IR) absorption spectrome-ter (Mattson Instruments, Inc., Galaxy 7020A). The powder sampleswere dried on a hot plate at 50 ◦C in a hood for one week tominimize the water content. To record the FT-IR absorption spec-tra (400–4000 cm−1), pellets of the powder samples in KBr wereprepared. The amount of surface coating was estimated with athermo-gravimetric analyzer (TGA) (TA Instruments, SDT-Q 600).The TGA curves of the powder samples were recorded between

room temperature and 700 ◦C while air flowed. The amounts ofPEGD, BSA, and C-BSA per GNP were estimated by recording the TGAcurves. Water desorption between room temperature and ∼110 ◦Cwas considered in these estimations.

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.6. Relaxivity and map image measurement

Both the longitudinal (T1) and transverse (T2) relaxation timesnd longitudinal (R1) and transverse (R2) map images of thequeous sample solutions of BSA-PEGD-GNPs and C-BSA-PEGD-NPs were measured using a 1.5 T magnetic resonance imaging

MRI) instrument (GE 1.5 T Signa Advantage, GE medical system)quipped with a Knee coil (EXTREM). Aqueous sample solutions atifferent Gd concentrations were prepared by diluting the origi-al sample solutions with triply distilled water. Both map imagesnd relaxation times were measured using these solutions. The lon-itudinal (r1) and transverse (r2) water proton relaxivities werestimated from the slopes in the plots of 1/T1 and 1/T2 versushe Gd concentration, respectively. The typical parameters used to

easure the relaxation times and map images were as follows:he external MR field (H) = 1.5 T, the temperature (T) = 22 ◦C, theumber of acquisition (NEX) = 1, the field of view (FOV) = 16 cm,he phase FOV = 1, the matrix size = 512 × 512, the slice thick-ess = 5 mm, the spacing gap = 0, the pixel bandwidth = 61.0547, theepetition time (TR) = 2009 ms, and the echo time (TE) = 9 ms.

.7. In vitro cytotoxicity measurement

The in vitro cytotoxicity of the aqueous sample solutions of

SA-PEGD-GNPs and C-BSA-PEGD-GNPs was measured using bothuman prostate cancer (DU145) and normal mouse hepatocyteNCTC1469) cells. A CellTiter-Glo Luminescent Cell Viability AssayPromega, WI, USA) was used to measure the cytotoxicity. In this

Fig. 1. HVEM images of (a) and (b) ultrasmall GNPs at two differe

ysicochem. Eng. Aspects 450 (2014) 67–75

assay, the intracellular ATP was quantified using a luminometer(Victor 3, Perkin Elmer). The cells were seeded onto a 24-well cellculture plate and incubated for 24 h (5 × 104 cell density, 500 �Lcells per well, 5% CO2, 37 ◦C). A series of test sample solutions (0,10, 100, and 200 �M) were prepared by diluting the original samplesolutions with a sterile phosphate buffer saline solution. ∼2 mL ofeach test solution was added to the cell culture media. The treatedcell culture media were then incubated for 48 h. The cell viability ofeach cell was determined and normalized with respect to that of thecontrol cell with 0.0 M Gd concentration. The measurements for alltest cells were repeated twice to obtain the average cell viabilities.

2.8. In vivo 3 T T2 MR image measurement

A 3 T MRI instrument (SIEMENS 3.0 T MAGNETOM Trio a Tim)was used to measure the T2 spin echo (SE) images of a mouse.The animal experiment in this study was carried out under thepermission and guidance of the KNU animal committee. An ICRfemale mouse (ICR—Institute of Cancer Research, USA) with aweight of ∼100 g was used for the MR image measurements.The mouse was anesthetized by 1.5% isoflurane in oxygen. Themeasurements were made before and after injecting the samplesolution into a mouse tail vein. The injection dose was ∼250 �L(∼0.1 mmol Gd kg−1). After the measurement, the mouse was

revived from anesthesia, placed into a cage, and given a free accessto both food and water. During the measurement, each mousewas maintained at ∼37 ◦C using a warm water blanket. The typ-ical measurement parameters were as follows: the H = 3 T, the

nt scales, (c) BSA-PEGD-GNPs, and (d) C-BSA-PEGD-GNPs.

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Md.W. Ahmad et al. / Colloids and Surfaces

= 37 ◦C, the NEX = 3–4, the FOV = 60 mm, the phase FOV = 30 mm,he matrix size = 128 × 256, the slice thickness = 1 mm, the spacingap = 0.1 mm, the TR = 2690 ms, and the TE = 37 ms.

. Results and discussion

.1. Particle diameter (d) and crystal structure of ultrasmall GNPs

Fig. 1a–d shows HVEM images of the as-prepared ultra-mall GNPs, BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs. The particleiameters of the ultrasmall GNPs ranged from 1 to 3 nm with theavg of 2.0 nm (Fig. 1a and b). The HVEM image of BSA-PEGD-GNPsndicated that many ultrasmall PEGD-GNPs were conjugated to aSA (Fig. 1c). On the other hand, the HVEM image of C-BSA-PEGD-NPs showed that many C-BSAs were conjugated to each ultrasmallEGD-GNP (Fig. 1d). As will be discussed later, this is consistentith the numbers of PEGD-GNPs conjugated to BSA and C-BSA

stimated from TGA analyses.The XRD patterns of powder samples of both BSA-PEGD-GNPs

nd C-BSA-PEGD-GNPs were measured before and after TGA andre provided in Fig. 2. The XRD patterns of the as-prepared pow-er samples were broad, due likely to ultrasmall particle diameters22]. On the other hand, the XRD patterns of the TGA-treated pow-er samples revealed sharp peaks, corresponding to monoclinicdPO4. All peaks after TGA analysis could be assigned to monoclinicdPO4 and the peak positions with sufficient intensities are markedith ‘*’ in Fig. 2 and the Miller index (h k l) assignments of these

eaks are provided in Supporting information. The formation ofdPO4 after TGA analysis is because the EDC/NHS coupling reactionas carried out in PBS. That is, the PO4

3− ions were likely attachedo amine groups of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs

20 40 60

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ig. 2. XRD patterns of powder samples of (a) BSA-PEGD-GNPs and (b) C-BSA-PEGD-NPs before (i.e. as-prepared) and after TGA analysis. All peaks after TGA analysisould be assigned to monoclinic GdPO4 and only the peak positions with suffi-ient intensities are marked with ‘*’ (Miller index (h k l) assignments are providedn Supporting information).

sicochem. Eng. Aspects 450 (2014) 67–75 71

through hydrogen bonding [23–25]. The estimated cell constantsare a = 6.644 A, b = 6.841 A, c = 6.328 A, and = 103.976◦ for TGA-treated BSA-PEGD-GNPs and a = 6.643 A, b = 6.839 A, c = 6.326 A, and

= 104.001◦ for TGA-treated C-BSA-PEGD-GNPs which are consis-tent with the values reported in PCPDFWIN [26].

3.2. Surface modification of ultrasmall GNPs

The surface coating of ultrasmall GNPs with PEGD followed byconjugation to BSA or C-BSA was investigated by FT-IR absorptionspectroscopy. As mentioned previously, one group among the two–COOH groups in each PEGD was conjugated to an ultrasmall GNPand the other was left free for amide bonding to BSA or C-BSA.These were confirmed from the two different C=O stretchingvibrations in the FT-IR absorption spectrum of the PEGD-GNPs(Fig. 3a). The free –COOH was observed at 1730 cm−1 but the–COOH bonded to GNPs, at 1620 cm−1. The peaks at 2910 cm−1

(C–H stretch) and 1110 cm−1 (C–O stretch) also confirmed that thePEGDs were bonded to ultrasmall GNPs. The peak at 3420 cm−1

in the PEGD-GNPs was assigned to the water –OH stretch. The∼110 cm−1 red shift of the C=O stretch after bonding to GNPsfrom that of the free –COOH had been observed in a range ofthe metal oxide NPs coated with the ligands with –COOH groups[22,27–30], supporting this result. The successful amide bondformation between PEGD-GNPs and BSA (or C-BSA) was confirmedfrom the disappearance of a free C=O stretch at 1730 cm−1 in bothBSA-PEGD-GNPs and C-BSA-PEGD-GNPs (Fig. 3b). Instead, the N–Hstretch at 3440 cm−1 (overlapped with water –OH stretch) and theN–H bend at 1530 cm−1 were observed [31–33]. The reduced inten-

sity in N–H bend in both BSA-PEGD-GNPs and C-BSA-PEGD-GNPswas, however, observed owing to the amide bonding of aminegroups of BSA and C-BSA with PEGD-GNPs and the hydrogenbonding of amine groups of BSA and C-BSA with PO4

3− ions.

4000 300 0 200 0 100 0

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Waven umber (c m-1)

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BSA-PEGD- GNP

BSA

Wavenu mber (cm-1)

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3440

1620 1530

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2910

Fig. 3. FT-IR absorption spectra of powder samples of (a) PEGD and PEGD-GNPs and(b) BSA, BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs.

72 Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75

0 10 0 20 0 30 0 40 0 50 0 60 0 70 00

20

40

60

80

100 Water desorption = 9.8%

Temperatu re (oC)

Wei

ght (

%) 28.0%

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0 10 0 20 0 30 0 400 500 60 0 70 00

20

40

60

80

100

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Temperature (oC)

Wei

ght (

%)

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0 10 0 20 0 30 0 400 500 60 0 70 00

20

40

60

80

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Tempe rature (oC)

Wei

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r2 = 22.0 s-1mM-1

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Concent rat ion Gd (m M)

ig. 4. TGA curves of powder samples of (a) PEGD-GNPs, (b) BSA-PEGD-GNPs, andc) C-BSA-PEGD-GNPs.

To determine the amounts of BSA in the BSA-PEGD-GNPs and-BSA in the C-BSA-PEGD-GNPs, the TGA curves of PEGD-GNPs,SA-PEGD-GNPs, and C-BSA-PEGD-GNPs were recorded (Fig. 4a–c).ater desorption between room temperature and ∼110 ◦C was

onsidered in these estimations. The amount of PEGD was esti-ated to be 28.0% from the TGA curve of PEGD-GNPs (Fig. 4a).

he amounts of BSA-PEGD and C-BSA-PEGD were estimated to be7.4 and 61.7% from the TGA curves of BSA-PEGD-GNPs and C-SA-PEGD-GNPs, respectively (Fig. 4b and c). The amounts of BSAnd C-BSA were estimated to be 19.4 and 33.7% by subtractinghe amount of PEGD from those of PEGD-BSA and C-BSA-PEGD,espectively. Using the davg of 2.0 nm for the ultrasmall GNPs esti-ated from the HVEM image and assuming that their density is

he same as that (=7.407 g mL−1) [34] of bulk Gd2O3, the num-er of GNPs conjugated to each BSA and C-BSA were estimatedo be 8.8 and 0.2, respectively, which were consistent with HVEMbservations (Fig. 1b and c). Therefore, BSA is a good nanoparti-le carrier, but C-BSA is not. This can be explained using the sizesnd masses of BSA and C-BSA. That is, the mass of ultrasmall GNPsith the davg = 2.0 nm was estimated to be 10–20 kD by calculating

he volume of the ultrasmall GNPs and using the bulk density ofd2O3 [34], which are smaller than those of BSA (mass = 66.5 kDnd size = 14 × 4 × 4 nm). On the other hand, C-BSAs with massesf 6.67 and 2.01 kD estimated from GPC have sizes of 6.5 × 1.9 × 1.9

Fig. 5. Plots of 1/T1 and 1/T2 of the aqueous sample solutions of (a) BSA-PEGD-GNPs and (b) C-BSA-PEGD-GNPs as a function of the Gd concentration. The slopescorrespond to the r1 and r2 values, respectively.

and 4.4 × 1.2 × 1.2, respectively, assuming a cube root dependenceof the size on the mass. Therefore, C-BSA is not large enough inmass and size to bind many ultrasmall GNPs, which is similar tothe polymers.

3.3. Suggested structures of BSA-PEGD-GNP andC-BSA-PEGD-GNP

As mentioned before, the conjugation between PEGD-GNP andBSA (or C-BSA) is an amide bond between –COOH of PEGD-GNPand –NH2 of BSA (or C-BSA). The BSA consists of 607 aminoacids and has amino acids with a free –NH2 [35,36] that canbe used for the amide bonding to PEGD-GNP. In fact, 60 lysineswith a free –NH2 are in BSA. Therefore, there are plenty of free–NH2 in BSA which can be conjugated to PEGD-GNPs through theamide bonding. As described previously, ∼9 PEGD-GNPs were esti-mated to be conjugated to each BSA whereas ∼0.2 PEGD-GNPs,to each C-BSA. Based on these results, structures of the BSA-PEGD-GNP and C-BSA-PEGD-GNP were schematically drawn inScheme 1c.

3.4. Relaxivities and map images

Magnetic properties of gadolinium oxide nanoparticles havebeen well characterized [37]. They are paramagnetic but havean appreciable magnetic moment at room temperature. This isbecause Gd(III) has seven unpaired 4f-electrons (8S7/2). Therefore,appreciable r1 and r2 values are expected from sample solutions,which were in fact observed in this study. The r1 and r2 val-ues of BSA-PEGD-GNPs were estimated to be 6.0 s−1 mM−1 and28.0 s−1 mM−1, respectively, from the slopes in the plot of 1/T1 and

1/T2, as a function of the Gd concentration (Fig. 5a). In the sameway, the r1 and r2 values of C-BSA-PEGD-GNPs were estimated to be7.6 s−1 mM−1 and 22.0 s−1 mM−1, respectively (Fig. 5b). These val-ues are also listed in Table 1 along with those of the other chemicals

Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75 73

Table 1Water proton relaxivity (r1 and r2)a of various chemicals.

Chemical d or davgb Nc r1 r2 Hd Te Ref.

Gd-DTPA – – 4.1 – 0.47 38 [5]BSA-GNP 20–40 – 6.7 38.5 4.7 37 [10]BSA-PEGD-GNP 2.0 8.8 6.0 28.0 1.5 22 This workC-BSA-PEGD-GNP 2.0 0.21 7.6 22.0 1.5 22 This work

a Unit: s−1 mM−1.b Particle diameter or average particle diameter (nm).

fPiifactTaG(hiBatPiNc

Fa

3.6. In vivo 3 T T2 MR images of a mouse

c Number of GNPs conjugated to a BSA or C-BSA.d Applied MR field (T).e Sample solution temperature (◦C).

or comparison. The r1 and r2 values of BSA-PEGD-GNPs and C-BSA-EGD-GNPs are larger than those [5,6] of molecular Gd-DTPA. Thesencreased relaxivities were attributed to the high density of Gd(III)n the NPs. These larger values generally lead to a higher sensitivityor detecting diseases in the body through contrast enhancementsnd can also provide the same quality MR images as those of the Gd-helates at reduced doses. The r2 values are significantly larger thanhat of molecular Gd-DTPA, which is why only NPs are eligible as2 MRI contrast agents, whereas molecular agents are only suitables T1 MRI contrast agents. The r1 and r2 values of BSA-PEGD-NPs and C-BSA-PEGD-GNPs were similar to those [10] of BSA-GNP

d = 20–40 nm) measured at a higher applied MR field. On the otherand, considering that the water proton relaxivities increase with

ncreasing applied MR field, those of the BSA-PEGD-GNPs and C-SA-PEGD-GNPs will be larger than those of BSA-GNP at the samepplied MR field. This is due likely to the particle size effect ofhe GNP. Both aqueous solutions of BSA-PEGD-GNPs and C-BSA-EGD-GNPs showed clear dose-dependent contrast enhancementsn their R1 and R2 map images (Fig. 6a and b), suggesting that these

Ps are potential candidates for MRI contrast agents, which wereonfirmed in a mouse experiment.

ig. 6. R1 and R2 map images of aqueous sample solutions of (a) BSA-PEGD-GNPsnd (b) C-BSA-PEGD-GNPs as a function of the Gd concentration.

3.5. In vitro cytotoxicity

The in vitro cytotoxicity of the aqueous sample solutions of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs were measured using DU145and NCTC1469 cells with Gd concentrations up to 200 �M (Fig. 7aand b). The results showed that C-BSA-PEGD-GNPs were slightlyless toxic than BSA-PEGD-GNPs. This is probably because many C-BSA like polymers encapsulated the PEG-GNPs, as shown in theHVEM image (Fig. 1c), whereas many PEGD-GNPs were conju-gated to each BSA on the surface of BSA, as shown in the HVEMimage (Fig. 1b). Therefore, PEGD-GNPs were better protected in C-BSA-PEGD-GNPs than in BSA-PEGD-GNPs. The cell viability of bothsamples decreased gradually with increasing Gd concentration. Thecell viability of C-BSA-PEGD-GNPs at 100 �M Gd reached more than70% for both cells, whereas that of BSA-PEGD-GNPs reached ∼60%for both cells. These levels of cellular toxicity are sufficiently low tocarry out in vivo MRI experiments.

Because BSA could bind many ultrasmall Gd2O3 NPs, whereasC-BSA did not, as measured by TGA, an in vivo MRI experiment

0

20

40

60

80

100

120(a) DU145

NCTC146 9

Conce ntration Gd ( M) 0 10 100 20 0

Cel

l Via

bilit

y (%

)

0

20

40

60

80

100

120

Conc entration Gd ( M)

Cel

l Via

bilit

y (%

)

0 10 100 200

DU145NCTC146 9

(b)

Fig. 7. In vitro cytotoxicity of aqueous sample solutions of (a) BSA-PEGD-GNPs and(b) C-BSA-PEGD-GNPs using both DU145 and NCTC1469 cells.

74 Md.W. Ahmad et al. / Colloids and Surfaces A: Ph

Fig. 8. (a) 3 T T2 MR images of the liver of a mouse before and after injecting anao“

wPawn(iwtetlti2etPf

4

(tGao

(

[

[

[

queous sample solution of BSA-PEGD-GNPs into a mouse tail vein and (b) the plotf signal intensity in T2 MR images as a function of time after injection (0 indicatesbefore injection”).

as further carried out using an aqueous sample solution of BSA-EGD-GNPs. Although GNPs are generally used as T1 MRI contrastgents, the T2 MR images were investigated because the r2 valueas a lot larger than the r1 value, due to the appreciable mag-etization of ultrasmall GNPs at room temperature [37]. 250 �L0.1 mmol Gd/kg) of an aqueous solution of BSA-PEGD-GNPs wasnjected into a mouse tail vein and 3 T T2 MR images of the liver

ere taken before and after injecting the aqueous sample solu-ion. As shown in Fig. 8a, appreciable negative (or darker) contrastnhancements were observed in the mouse liver after the injec-ion, which returned to almost the original contrast after 24 h dueikely to the excretion of BSA-PEGD-GNPs. To more clearly see theime evolution of the contrast change in T2 MR images, the signalntensity in T2 MR images was plotted as a function of time up to4 h in Fig. 8b. This plot clearly shows that the negative contrastnhancement maintained up to 91 min after injection but returnedo almost zero above 91 min due likely to the excretion of BSA-EGD-GNPs. These results clearly indicate that the sample solutionunctioned as a T2 MRI contrast agent.

. Conclusions

In summary, we synthesized PEGD coated ultrasmall Gd2O3 NPsi.e. PEGD-GNPs) which were then conjugated to BSA and C-BSAhrough amide bonding (i.e. BSA-PEGD-GNPs and C-BSA-PEGD-NPs). We characterized physical and in vitro MRI properties,nd cytotoxicity of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs, andbtained in vivo MR images using BSA-PEGD-GNPs.

1) BSA (Mn = 66.5 kD) could bind many ultrasmall PEGD-GNPs(the core davg = 2.0 nm), showing that BSA is a good ultrasmallNP carrier. The TGA showed that ∼9 ultrasmall PEGD-GNPs

could be conjugated to each BSA. However, C-BSAs (Mn < 7 kD)could not bind many ultrasmall PEGD-GNPs due to its reducedsize and mass. Instead many C-BSAs were conjugated to anultrasmall PEGD-GNP like polymers. The TGA showed that ∼5C-BSAs were conjugated to each ultrasmall PEGD-GNP.

[

ysicochem. Eng. Aspects 450 (2014) 67–75

(2) MR relaxivity measurements revealed that both BSA-PEGD-GNPs and C-BSA-PEGD-GNPs had r1 and r2 values larger thanthose of molecular Gd-chelates.

(3) The 3 T T2 MR images after injecting an aqueous sample solu-tion of BSA-PEGD-GNPs into the mouse tail vein showed clearnegative contrast enhancements.

(4) Large relaxivities and in vivo T2 MR images prove that BSA-PEGD-GNPs and C-BSA-PEGD-GNPs are potential MRI contrastagents.

These results suggest that biological molecules such as BSA canbe used to conjugate many surface modified ultrasmall NPs whichcan be applied to a variety of biomedical areas such as MRI contrastagents studied in this work.

Acknowledgments

This study was supported by the Basic Science Research Pro-gram (Grant no. 2012R1A1B3004241 to KSC, 2011-0015353 to YC,and 2013R1A1A4A03004511 to GHL) and the Basic Research Labo-ratory (BRL) program (Grant no. 2013R1A4A1069507) through theNational Research Foundation funded by the Ministry of Education,Science, and Technology, the R&D program of MKE/KEIT (Grant no.10040393, development and commercialization of molecular diag-nostic technologies for lung cancer through clinical validation), andthe KNU Research Fund (2013). The authors wish to thank the KoreaBasic Science Institute for the use of their HVEM and XRD.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2014.03.011.

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